Analysis of the nucleotide sequence of the human genome, said to comprise as many as three billion base pairs, is progressing. In particular, the analysis of polymorphism in the human genome relates to the unique traits of each individual and is the subject of considerable interest in the fields of medicine, pharmacology, and biology. Conventionally, the development of a sequencer capable of automatically processing multiple samples simultaneously, rapidly, and with high sensitivity has been conducted to determine the huge nucleotide sequence of the human genome. In particular, the arrival of multicapillary DNA sequencers simultaneously employing multiple capillary columns packed with gel instead of the tabular slab gel that was previously employed has greatly contributed to increasing the speed of nucleotide sequencing. Currently, 96 capillary column (for example, the Sequencer 3700 from ABI and the MegaBACE 1000 from Molecular Dynamics), and recently, the fourfold 384 capillary column (the development of the 384 multicapillary sequencing system: Proceedings of the 21st Meeting of the Japan Molecular Biology Society, 1P-570 (Yokohama, December 1998) multicapillary DNA sequencers have been developed.
However, when the number of capillary columns is increased, there are limitations due to the performance and structure of the detection devices used to read the sequences. Further, merely increasing the number of capillary columns does not diminish the labor required to supply the capillary columns themselves and to load specimens into capillary columns.
Further, in currently employed sequencers, about 500 bases are read by a single capillary column. Increasing the number of bases read by a single capillary column to increase separation performance has the advantage of reducing the effort required to supply the capillary columns themselves and to load specimens into the capillary columns. However, almost no studies have been conducted into reading more bases at once in individual capillary columns; this remains a problem to be solved in the future.
For example, in many electrophoretic devices including DNA sequencing devices, a support member is employed to support the electrophoretic matrix. The support member has various shapes depending on the electrophoretic objective. For example, in slab electrophoresis, two pieces of flat tabular silica glass are employed. In capillary electrophoresis, a column-shaped (hollow tubular) silicate capillary column is employed as the support member. Further, in micro electrophoresis, a support member with minute separation passages formed on a silicon wafer is employed. All of these support members are generally made of silicon-containing materials, such as silica.
To obtain good separation performance in electrophoretic matrices prepared on such silicon-containing support members (referred to hereinafter as simply “support members”), it is desirable for bubbles not to be present in the matrix.
For example, when electrophoretic gel is sandwiched between two support members (in slab electrophoresis), when an entangled polymer solution is injected into a cavity in a support member (capillary electrophoresis), or when a support member is employed without being cleaned, particularly when the width of the support member or injection inlet are narrow (when the width is less than about 1 mm in diameter), air bubbles tend to develop near the surface and in the minute voids of the support member. Gel and entangled polymer matrices containing such bubbles do not provide adequate separation performance and cannot perform the original functions of electrophoretic matrices.
The method of admixing propanol or polyethylene glycol in advance to polymerization has been proposed as a method of preventing the development of such bubbles (Anal. Chem., 1992, 64, pp. 2665-2671; J. Chromatogr., 1991, 550, pp. 823-830). This method yields a matrix that does not contain bubbles. However, the support obtained by this method has the drawback of having lower separation performance in electrophoresis than conventional supports. Thus, this method is unsuitable for electrophoresis requiring high separation performance.
Further, assuming that impurities present on the surface of the support member are the cause of the bubbles, attempts at inhibiting the occurrence of bubbles have been made by the method of cleaning away impurities on the inner walls of capillary columns using a strong base solution such as NaOH, organic solvents such as ethanol, acid solutions such as HCl, and solutions of these compounds in combination by using pure water (Electrophoresis 1996, 17, pp. 144-151). However, although this method prevents the occurrence of bubbles at the surface of the support member, electrophoretic separation performance is unsatisfactory in the same manner as in the above-described method.
Accordingly, there has been for some time a need to provide a method of preparing electrophoretic supports capable of effectively inhibiting the occurrence of bubbles in matrices carried by or packed in support members that deliver good separation performance.
Further, in addition to methods of preparing electrophoretic supports, there has also been room for improvement in the electrophoretic matrix itself to improve electrophoretic separation performance, However, there has not been adequate investigation of electrophoretic matrix materials and the like.
Accordingly, the object of the present invention is to provide a means of solving the above-stated problems in electrophoresis, improving electrophoretic separation performance, improving sample separation, and particularly, in nucleic acid and PNA fragments, reading even longer-strand nucleotide sequences.
More specifically, the object of the present invention is to provide a method of preparing an electrophoretic support capable of fin inhibiting the generation of bubbles in the matrix when preparing an electrophoretic support employing a silicon-containing support member, and to provide a method of electrophoresis employing the electrophoretic support prepared by this method.
A further object of the present invention is to provide an electrophoretic gel tending not to undergo compression in the nucleotide sequencing of long-strands exceeding 500 bases, and to provide a method of electrophoresis capable of reading long-strand nucleotide sequences using this gel.